Obesity predisposes humans to diabetes and cardiovascular disease and is a universal threat to health. Fat storage is dynamically controlled by orchestrated hormonal, neural and metabolic signals between brain and periphery. Proopiomelanocortin (POMC) neurons that synthesize melanocortin peptides are a primary integrative site for these diverse signals related to energy homeostasis. In the past funding cycles, we made significant progress towards explaining how Pomc transcription is restricted to a subset of hypothalamic neurons. A modular locus comprised of two evolutionarily distinct enhancers directs neuron-specific Pomc expression. Targeted deletion of the individual nPE1, nPE2 or combined elements revealed that they act synergistically during hypothalamic development and additively in adult life to maintain sufficiently robust Pomc transcription to avoid obesity. A bioinformatic analysis of core nucleotide sequence motifs within the enhancers combined with anatomic and functional interrogation of candidate homeodomain transcription factors (TFs) in animal models identified a major contribution of Isl1 and Nkx2.1 in directing the unique temporal and spatial patterns of Pomc expression in the arcuate nucleus. Although necessary, these two factors alone are not sufficient to fully account for the complexities of neuronal Pomc regulation. Therefore, we propose the following specific aims for this project renewal: 1) Decipher the transcriptional code that controls hypothalamic Pomc expression through the functional identification of the entire set of TFs that control the embryonic development, identity and maintenance of the POMC neuronal phenotype using complementary mouse and zebrafish molecular genetics; 2) Identify the entire set of cis-acting regulatory elements in the neuronal enhancers that control hypothalamic Pomc expression and investigate their interactions to assemble a fully functional transcriptional locus; and 3) Dissect the physiological roles of nPE1, nPE2, and their cognate TFs on Pomc expression and metabolic regulation associated with leptin signaling and the energy demands of pregnancy and lactation. These studies will provide fundamental knowledge about a gene essential for regulating body mass and possibly identify novel genetic or signaling pathways that can be exploited for therapeutic purposes.